The present invention relates in general to an automatic rate analyzing method and in particular concerns an improvement of an automatic rate analyzing system with a view to enhancing a sample processing speed.
Lately, reaction rate measuring methods (hereinafter referred to also as rate method in abridgment) have been increasingly adopted in field of the clinical biochemical analysis. The rate method is in principle advantageous over the hitherto known colorimetric analysis in that relatively high accuracy of measured values can be attained with the endogenous error being suppressed to a minimum. However, the rate method is inferior to the colorimetric analysis in respect of the time required for processing a sample because examination or observation of the individual sample to be analyzed takes a relatively long time. In spite of the fact that many and various attempts have been made in an effort to increase the processing speed of various automatic analyzing apparatus, there is still unavailable a system which can satisfy the desire and demand of users in respect of both the accuracy or precision of measurements and the speed at which the sample to be examined is processed.
As the means for increasing the processing speed of the rate analysis in the automatic analyzing apparatus the following measures are conceivable:
(1) Improving the stability and resolution of a photometer as used (e.g. by lowering the lower limit of analysis). PA0 (2) Decreasing the dilution ratio (the ratio in quantity between a sampled serum and a final solution). PA0 (3) Simultaneous measurement of plural samples in parallel. PA0 (4) Development of high sensitive reaction series and reagents.
In conjunction with the first mentioned photometer, studies and developments have been made on the diffraction grating, high energy light source, semiconductor sensor and the like. At present, the stability on the order of 1.times.10.sup.-4 OD (optical density, absorbance) has been attained.
When the dilution is decreased, i.e. part of serum is increased, the sensitivity of the substance to be analyzed (variation in absorbance) is correspondingly increased, thereby to allow the rate measurement to be carried out in a short time. However, the accuracy which is one of the most advantageous features of the rate analysis undergoes degradation, whereby the range of measurement is disadvantageously restricted. For this reason, the reduction of the dilution is unfavorable. Practically, the ratio of dilution is preferably in the range of 30 to 60 in the conventional automatic analyzer apparatus. Lately, an automatic analyzing apparatus of a discrete type has been commercially available which is capable of accomplishing the reaction rate measurement of a single sample for about twenty seconds by using the dilution ratio on the order of 10, thereby to permit the processing to be carried out at a speed of 120 samples/hour. However, there is a problem remaining to be solved in respect of the attainable accuracy.
As to the parallel measurements of plural samples, there is typically known as apparatus called the Anderson's centrifugal system. With this apparatus, it is certainly possible to process several tens of samples for several minutes so far as the photometry is concerned. However, due to the rotary system as adopted, there arise difficulties in respect of linkage to other analyzing operations such as pippetting, dispensing, incubation, washing or the like. Additionally, it is impossible to carry out measurements simultaneously on plural items. After all, the processing speed can not be increased as a whole to a desired degree.
Development of new reaction series and reagents is certainly one of the most important problems to be solved, which however requires intrinsically a train of repeated try-and-error processes and is not adapted to a straightforward solution.
Notwithstanding the circumstances described above, there is at present a pressing demand for the automatic rate analyzer apparatus of the discrete type which can be operated in a facilitated manner at an increased processing speed with an enhanced accuracy and precision. One advantageous feature of the discrete type automatic rate analyzing apparatus resides in that a number of items can be simultaneously and continuously measured in a set of reaction series. On the other hand, this type of analyzing apparatus has a drawback in that the operation speed of the whole system has to follow the processing speed at the photometrical station because the individual chemical controls and manipulations including measurements are carried out on a continuous line base. A typical example of such automatic rate analyzing apparatus is disclosed in U.S. patent application Ser. No. 834,902 filed Sept. 20, 1977 by K. Yamashita, H. Umetsu, K. Heguri and K. Yoshida under the title "Automatic Chemical Analyzing Method and Apparatus" and assigned to the assignee of the present application, which application corresponds to Japanese patent application No. 51-114226(1976) filed Sept. 22, 1976 by the present assignee under the title "Automatic Chemical Analyzing Apparatus" and disclosed publicly Apr. 10, 1978 as Japanese Laid-Open patent application No. 53-39192(1978).
In the case of the automatic rate analyzing apparatus disclosed in the patent applications cited above, a number of samples or specimens (sera) each added with reagent are discretely transported on a reaction line. The samples in which chemical reactions have been brought about are individually irradiated by light radiation during the transportation, whereby the quantity of light of a specified wavelength transmitted through the individual sample is measured by a spectrophotometer twice at different time points. Then, the absorbances at the different time points are arithmetically determined on the basis of the measured values through logarithmic calculation. From the difference in the calculated absorbances, variation in the absorbance of the individual sample during the predetermined time is arithmetically determined and used to represent the concentration or activity of the concerned items in the serum. In the rate analyzing apparatus described above in which the magnitude of variation in the absorbance is determined in terms of the difference in absorbance at two different time points, i.e. at the beginning and the end of the photometery period, there arises a problem that the stability of the photometer system becomes degraded due to possible noise, particularly when the number of samples is small. Further, in order to detect the activity of the item in concern with a reasonable accuracy, the period during which the sample to be analyzed is examined by the photometer system has to be correspondingly elongated, as the result of which the processing speed is necessarily decreased. In this respect, the automatic rate analyzing apparatus is inferior to the colorimetric analysis. Besides, the logarithmic calculation for determining the absorbance on the basis of the quantity of light transmitted through the sample will take a lot of time when the calculation is executed by using a micro-computer, providing an obstacle in increasing the processing speed. In this manner, the automatic rate analyzing apparatus described above has many problems remaining to be solved for satisfying the requirements imposed by the users in respect of the accuracy and precision as well as the sample processing speed.
The stability of the conventional photometer is on the order of 10.sup.-4 OD as described hereinbefore. On the other hand, in the popular rate measurement of enzymes GOT (glutamate oxalate transaminase) and GPT (glutamate pyruvate transaminase) in which the absorbance change of the reagent NADH is measured, the change or variation in absorbance for one international unit (1 mU/ml) is on the order of 1.times.10.sup.-4 OD/mm at the dilution factor of 50. Accordingly, the time duration required for the photometry of one sample will amount to about 30 minutes in order to attain the rate measurement with a high accuracy, which in turn means that the processing speed of the analyzing apparatus as a whole is on the order of 60 samples/hour. Such processing speed is of course remarkably lower as compared with the processing speed in the conventional colorimetric analysis. There is certainly available a discrete type analyzing apparatus which is capable of processing 120 samples an hour by using a decreased dilution ratio. Such apparatus however is disadvantageous in respect of accuracy and allowable range of measurement, as described hereinbefore.
It should be mentioned that, in the automatic analyzing apparatus of the discrete type, the chemical controls or manipulations other than the photometry such as pippetting, dispensing, agitation, washing or the like can be carried out at a speed of about 10 seconds for a sample. Accordingly, when the processing speed at the photometric system is increased, a high speed automatic rate analyzing apparatus can be realized which permits a single sample to be processed within 10 seconds, i.e. an increased processing speed of about 360 samples for an hour.
In the case where one cycle is to be completed within 10 seconds, the time duration required for the rate photometry has to be in the range of 5 to 8 seconds, with the result that a variation or change in absorbance for the measurement of GOT and GPT becomes remarkably small on the order of 1.2.times.10.sup.-5 OD to 8.times.10.sup.-6 OD. The stability of the photometric system should then be about 1.times.10.sup.-5 OD.
It should be additionally mentioned that, in the measurement of such minute change in absorbance as described above, the resolution of an A-D (analog-to-digital) converter used in the arithmetic operation brings about a problem in addition to the problem of stability. The problem of the resolution of A-D converter is serious in consideration of the fact that most of the rate reactions take place in a high range of absolute value of absorbance (e.g. 10 to 2.0 OD). The resolution of the commercially available A-D converter is at most on the order of 16 bits (in the binary system). Accordingly, the output of the A-D converter is about 2000 in decimal notation which means that the resolution power of the A-D converter represented in terms of absorbance is remarkably low as on the order of 2.times.10.sup.-4 OD.
For the determination of the absorbance change with a view to enhancing the stability of the photometric system inclusive of the succeeding data processing parts, the absorbance is measured 2m+1 times and a regression line is determined through the least square approximation. Then, the absorbance change .DELTA.ABS over the whole photometry period is determined on the basis of the slope of the regression line. Namely, ##EQU1## where x represents time points obtained by dividing the measuring period by 2m+1, and Ax thus represents the absorbance at the time point x. The stability of the photometric system inclusive of the data processing parts can be enhanced through the procedures described above. In this connection, an increased number of data samplings brings about a correspondingly improved stability. In general, assuming that noise components are present in the Gaussian distribution, the stability, of the whole photometric system becomes substantially 1/.sqroot.m after the procedure described above. However, when the above arithmetic procedure is to be executed by a computer, then there arises a problem in connection with the processing speed of the employed computer. This problem is serious particularly when the arithmetic processing is to be executed by a micro-computer adopted usually in the automatic rate analyzing apparatus. In the determination of the absorbance change on the basis of the mathematic expression (1), the time consuming process is the logarithmic computation for determining the absorbance A from the transmissivity T. For example, if the calculation based on the equation (1) is to be executed by the conventional micro-computer, then 70 to 80% of the whole calculation time will be consumed for the logarithmic operation.
As will be appreciated from the foregoing discussion, there has not been known yet a rate analyzing method which can assure high accuracy and precision of measurement at a high processing speed.